Analysis of ColE1 MbeC Unveils an Extended Ribbon-Helix-Helix Family of Nicking Accessory Proteins

Abstract

MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.

MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

“…The presence of the highly conserved structural motif LxxugxNlNQiaxxLn in the C- terminal part classifies MobC and its PromA homologs [44–46], as the putative conjugative transfer auxiliary proteins of MOB P relaxases [13]. …”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Recent studies have identified a family of Dtr accessory factors with a common structural basis for oriT binding and relaxase recruitment. These factors possess a ribbon-helix-helix (RHH) DNA binding motif resembling that described previously for the Arc/ CopG/Mnt family of transcriptional regulators (36,268,291 (36,58,192,268,291,297,299). X-ray structures are now available for RHH domains of NikA and VirC2, and results of structurebased mutational analyses firmly establish the importance of the RHH domain in oriT binding as well as the recruitment of and nicking by the relaxase (179,291).…”

Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria

“…The tra genes encode a relaxase, a mating pair formation (MPF) complex and a type IV coupling protein. The oriT is recognized by the relaxase, which binds and nicks the DNA at the nic site within the oriT (Varsaki et al, 2009). The relaxase together with one or several auxiliary proteins are termed the relaxosome (Pansegrau et al, 1990).…”